This project, in part, represents an extension of work previously reported as Project Numbers Z01 DK69037, Z01 DK069097 and Z01 DK069000. It also reports on continuation of work previously reported under Project Numbers DK069036-23, DK069063-17, and DK069100-06. All work related to diabetic nephropathy, with the exception of the genetics of diabetic nephropathy, is now reported under this single project. In the last year, we continued to work with an international consortium known as the CKD Prognosis Consortium that is examining the prognostic implications of low estimated glomerular filtration rate and elevated albuminuria for mortality, kidney disease, and cardiovascular outcomes. This collaboration resulted in five additional publications this year, including two in Lancet, one in the Journal of the American Medical Association, one in the British Medical Journal, and one in the International Journal of Epidemiology. Four of these papers describe associations of kidney disease measures with the pre-specified health outcomes according to the presence or absence of diabetes, hypertension, age, and sex. They each emphasize the importance of kidney disease per se as a predictor of health outcomes. The fifth is a summary description of the more than 50 cohorts that make up this consortium. In the coming year, this consortium will look at other predictors of cardiovascular disease and end-stage renal disease in the cohorts, as well as the impact of chronic kidney disease progression on end-stage renal disease and mortality. We also published three papers in Lancet and we have one in press in the Journal of the American Medical Association as part of the Global Burden of Disease Collaboration. Our contribution to this collaboration was to assist in the development of global and regional disease burden estimates related to the complications of diabetes. In the coming year, this collaboration will work to update Global Burden of Disease estimates, with the goal of making these updates annually. A major finding of the collaboration is the move away from infectious disease and towards chronic disease as a major source of disease burden worldwide, with a growing burden of disability as fewer people die early because of these diseases, but instead increasingly become disabled. Our single-center clinical trial, which was a randomized, double-blinded, placebo-controlled study involving 170 adults with type 2 diabetes and normal urinary albumin excretion or microalbuminuria was recently published in Diabetes. It was designed to determine whether blockade of the angiotensin II receptor with losartan would prevent or further attenuate the development and progression of early diabetic nephropathy in subjects with type 2 diabetes mellitus who were receiving standard diabetes care. Subjects in each albumin excretion group were randomized to treatment with either losartan, or placebo. Measurements of glomerular filtration rate were made using a standard urinary clearance method, with iothalamate as the filtration marker. These tests were performed initiially, at one month, and at 12-month intervals from baseline thereafter. The primary outcome measure was a decline in GFR to less than 60 ml/min or to half the baseline value in subjects that enter the study with a GFR of <120 ml/min. A kidney biopsy was performed after 6 years in 111 of these subjects. Morphometric analysis of kidney biopsies was used to determine differences in glomerular structure between treatment groups. We found that expansion of mesangial fractional volume, a structural hallmark of diabetic nephropathy that is strongly associated with depression of the glomerular filtration rate, was significantly attenuated in the group that received losartan. This phenomenon was accompanied by higher filtration surface density and area compared with placebo, a finding that would enhance glomerular filtration capacity. We alsoo observed a 51% reduction in risk of GFR decline in losartan-treated subjects compared with placebo, and a 46% reduction in risk of progression to macroalbuminuria in those with microalbuminuria at baseline, although neither of these differences in functional outcomes was statistically significant. Nevertheless, our findings suggest that early treatment with losartan in those with microalbuminuria may preserve glomerular architecture, possibly providing a structural basis for the renoprotective effect of angiotensin receptor blockade seen previously. In a paper published in the American Journal of Kidney Diseases, we found that cystatin C, an endogenous low molecular mass protein that is produced at a constant rate and virtually completely cleared from the circulation by glomerular filtration with subsequent proximal tubular uptake and degradation, is a better predictor of progression to end-stage renal disease than measurement of either the glomerular filtration rate or the serum creatinine concentration among persons with type 2 diabetes and elevated albuminuria. In subjects with normal or high-normal glomerular filtration rate, the predictive ability of cystatin C remained superior to the other filtration markers, suggesting that measurement of cystatin C may allow earlier risk stratification of patients at high risk for progression to kidney failure. We concluded that the predictive value of cystatin C for end-stage renal disease in patients with type 2 diabetes may be enhanced beyond the measurement of glomerular filtration rate by the urinary clearance method, which is generally considered the gold standard, because of additional renal and non-renal information cystatin C may impart. Finally, in a paper published in Diabetes, we showed how functional genomics based hypothesis generation can be implemented by a stepwise integration of regulatory SNP prediction, transcriptional promoter modeling and pathway analysis. In another paper in the same journal, we also demonstrated that a transforming growth factor beta-induced feedback amplification circuit between p53 and microRNA-192 related to the pathogenesis of diabetic nephropathy in Pima Indians, and the microRNA-192 knockout mice are protected from key features of diabetic nephropathy. In a third paper in the same journal, using an assay in which normal human podocytes are cultured with the sera of patients with diabetic kidney disease, we showed that circulating factors other than glucose may directly affect podocyte function in vitro. We identified a novel mechanism in which intracellular cholesterol accumulation due to impaired efflux plays a central role in podocyte injury in vitro. These data suggest that this mechanism may contribute to the development and/or the progression of diabetic kidney disease. Furthermore, using cyclodextrin in our in vitro and in vivo studies, we were able to protect podocytes from injury caused by intracellular cholesterol accumulation, indicating that cyclodextrin may represent a safe and effective way to target reverse cholesterol transport in diabetes and diabetic kidney disease. In the coming year, our systems biology work will expand to evaluate changes in tissue-level gene expression that occur with progression of diabetic kidney disease. We will perform a second kidney biopsy on patients previously enrolled in the losartan clinical trial described above in our ongoing effort to characterize the molecular pathways responsible for progressive diabetic kidney disease.